Sphingolipid rheostat changes have been implicated in many neurodegenerative diseases, such as Alzheimer's disease, HIV-induced dementia and Parkinson's disease. These changes could be pathogenic events or homeostatic responses, but the underlying mechanisms are not clear. To further explore the roles of sphingolipids in neurodegeneration, I will take advantage of a mouse CerS1 (ceramide synthase 1) mutant, which displays early-onset Purkinje cell degeneration and adult-onset widespread accumulation of protein inclusions in neurons. Steady state levels of ceramide/sphingolipid, especially C18 ceramide/sphingolipid, are decreased in the mutant brain, whereas a striking increase in signaling-potent sphingoid bases and their phosphorylated metabolites, especially dihydrosphingosine (dhS) and dhS-1-phosphate (dhS1P), has been observed. The reduction of ceramide and complex sphingolipids may result in deficiency in membrane protein targeting which could lead to protein inclusions, and/or membrane organelle defects. On the other hand, drastic increase of sphingoid bases might interfere with normal sphingolipid-mediated signaling. Because very few Purkinje cells die with visible protein inclusions, this early pathological event is likely to be caused by changes in signaling function. In contrast, the late event, protein inclusion formation, may be due to impaired structural functions of sphingolipids. To find out whether the drastic increase of sphingoid bases impairs neuronal functions and causes neurodegeneration of Purkinje cells, and whether reduction of total ceramide or C18 ceramide contributes to Purkinje cell death and/or accumulation of protein inclusions in other neurons, I will: 1) Identify the preclinical intracellular changes that are potentially regulated by sphingoid lipids, particularly sphingoid bases. The initial study will focus on sphingosine-1-phosphate (S1P) signaling via its receptor S1PR1 (S1P1). Because dhS1P is also an agonist for S1P receptors, I propose that excessive dhS1P interferes with S1P1 signaling. 2) Test whether modulating sphingolipid profile in vivo will modify the CerS1 mutant phenotype. Modulation of sphingolipid profile will be accomplished by two methods, decreasing serine palmitoyltransferase (SPT) activity by using Sptlc1 heterozygosity or increasing ceramide synthesis by transgenic overexpression of CerS6. The result of decreasing SPT activity in CerS1 mutant background will determine if the cause of the pathological event in question is due to increased sphingoid bases or decreased ceramide level. The result of CerS6 overexpression will confirm the conclusion and further determine whether reduction of total ceramide or C18 ceramide species is the cause. PUBLIC HEALTH RELEVANCE: Changes of sphingolipid profiles have been observed in some neurodegenerative diseases in which the causal mutations do not have apparent functions in lipid metabolism, including Alzheimer's disease, Parkinson's disease and HIV-induced dementia. However, it is not clear whether these changes are pathological events or homeostatic responses. This timely application will directly address how sphingolipid homeostasis changes will impact on the health of the brain using a unique mouse model.